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Purification and Characterization of Phosphoglycerate Mutase from Methanol-grown Hyphomicrobium
x and Pseudomonas
am1
Abstract
SUMMARY: Phosphoglycerate mutase has been purified from methanol-grown Hyphomicrobium x and Pseudomonas ami by acid precipitation, heat treatment, ammonium sulphate fractionation, Sephadex G-50 gel filtration and DEAE-cellulose column chromatography. The purification attained using the Hyphomicrobium x extract was 72-fold, and using the Pseudomonas ami extract, 140-fold. The enzyme purity, as shown by analytical polyacrylamide gel electrophoresis, was 50% from Hyphomicrobium x and 40% from Pseudomonas ami. The enzyme activity was associated with one band. The purified preparations did not contain detectable amounts of phosphoglycerate kinase, phosphopyruvate hydratase, phosphoglycerate dehydrogenase or glycerate kinase activity. The molecular weight of the enzymic preparation was 32000 ± 3000. The enzyme from both organisms was stable at low temperatures and, in the presence of 2,3-diphosphoglyceric acid, could withstand exposure to high temperatures. The enzyme from Pseudomonas ami has a broad pH optimum at 70 to 76 whilst the enzyme from Hyphomicrobium x has an optimal activity at pH 73. The cofactor 2,3-diphosphoglyceric acid was required for maximum enzyme activity and high concentrations of 2-phosphoglyceric acid were inhibitory. The K m values for the Hyphomicrobium x enzyme were: 3-phosphoglyceric acid, 6·0 × 10−3 m; 2-phosphoglyceric acid, 69 × 10−4 m; 2,3-diphosphoglyceric acid, 80 × 10−6 m; and for the Pseudomonas ami enzyme: 34 × 10−3 m, 37 × 10−4 m and 10 × 10−6 m respectively. The equilibrium constant for the reaction was 11·3 ± 2·5 in the direction of 2-phosphoglyceric acid to 3-phosphoglyceric acid and 0·09 ± 0·02 in the reverse direction. The standard free energy for the reaction proceeding from 2-phosphoglyceric acid to 3-phosphoglyceric acid was −5·84 kJ mol−1 and in the reverse direction +5·81 kJ mol−1.
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© Society for General Microbiology, 1976